Genetic and functional analyses of the conserved C-terminal core domain of Escherichia coli FtsZ
Genetic and functional analyses of the conserved C-terminal core domain of Escherichia coli FtsZ
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DOI：
10.1128/jb.181.24.7531-7544.1999
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发表时间：
1999-12-01
影响因子：
3.2
通讯作者：
Margolin, W
中科院分区：
文献类型：
--
作者：
Ma, XL;Margolin, W
在Escherichia call中，FtsZ是必需的细胞分裂蛋白FTSA和ZipA重新聚集到隔环所必需的。E.call FtsZ的几个C-末端缺失，包括仅有的12个氨基酸中去除高度保守的C-末端核心区的一个，在表达于质粒上时未能补充染色体FtsZ突变体。为了识别核心区内的关键个体残基，6个高度保守的残基被丙氨酸取代，除了一个突变体(D373A)外，所有这些突变体(D373A)都未能补充StsZ染色体突变体。免疫印迹分析表明，虽然I374A和F377A蛋白在细胞内不稳定，但L372A、D373A、P375A和L378A蛋白的合成水平正常，表明它们在FtsZ功能的某些方面存在特异性缺陷。此外，所有四个稳定的突变蛋白都能够在染色体FtsZ突变的潜在分裂位点定位并形成环，这意味着除了定位和多聚体之外，还有一种功能缺陷。由于FtsZ的另一个被提出的功能是招募FTSA和ZipA，我们测试了C末端核心域是否对与这些蛋白质的相互作用重要。利用两种不同的体内实验，我们发现FtsZ的12个氨基酸截断在与FTSA的结合上存在缺陷。此外，该区域的两个点突变体(L372A和P375A)与FTSA的结合能力减弱，而ZipA能够与FtsZ C末端核心的所有四个稳定点突变体结合，但不能与12个氨基酸的缺失结合。
In Escherichia call, FtsZ is required for the recruitment of the essential cell division proteins FtsA and ZipA to the septal ring. Several C-terminal deletions of E. call FtsZ, including one of only 12 amino acids that removes the highly conserved C-terminal core domain, failed to complement chromosomal ftsZ mutants when expressed on a plasmid, To identify key individual residues within the core domain, six highly conserved residues were replaced with alanines, All but one of these mutants (D373A) failed to complement an StsZ chromosomal mutant. Immunoblot analysis demonstrated that whereas I374A and F377A proteins were unstable in the cell, L372A, D373A, P375A, and L378A proteins were synthesized at normal levels, suggesting that they were specifically defective in some aspect of FtsZ function. In addition, all four of the stable mutant proteins were able to localize and form rings at potential division sites in chromosomal ftsZ mutants, implying a defect in a function other than localization and multimerization. Because another proposed function of FtsZ is the recruitment of FtsA and ZipA, we tested whether the C-terminal core domain was important for interactions with these proteins. Using two different in vivo assays, we found that the 12-amino-acid truncation of FtsZ was defective in binding to FtsA. Furthermore, two point mutants in this region (L372A and P375A) showed weakened binding to FtsA, In contrast, ZipA was capable of binding to all four stable point mutants in the FtsZ C-terminal core but not to the 12-amino-acid deletion.